A research article in the September 2014 issue of the JCI identifies a microRNA-based strategy that inhibits arterial restenosis while preserving endothelial function. The authors created an adenoviral vector encoding the cyclin-dependent kinase inhibitor p27Kip1 with endothelial cell–specific microRNA target sequences that triggers cell cycle arrest in vascular smooth muscle cells without impairing endothelial cell proliferation that aids in healing. Their research provides proof of concept for a targeted microRNA-based method that reduces neointimal hyperplasia, accelerates re-endothelialization, and inhibits thrombosis in the setting of arterial injury. We asked the lead author, Gaetano Santulli, about his work.
What first motivated you to pursue this line of research?
MicroRNAs are tiny molecules of RNA that only a few years ago were considered not more than junk, since they are not translated into proteins as is conventional mRNA. Nevertheless, they have been recently implicated in numerous molecular processes, with functional roles in several human disorders (1). When Dr. Hana Totary-Jain, the senior author of the paper, first discussed this project with me, I thought that her idea of exploiting microRNA as a selective therapy to treat coronary artery disease was just brilliant. Since she was showing me the preliminary data in endothelial and smooth muscle cells, I was so enthusiastic and eager to work on it, especially given my training as a physician-scientist and cardiologist. I leveraged my expertise in microsurgery and in experimental ex vivo/in vitro assays in cardiovascular biology (2, 3). In order to test our strategy in vivo, we needed a large amount of vector, which was prepared by Anetta Wronska in our lab. Once we were able to obtain the first data in vivo, I felt a deep motivation: the results achieved day by day were simply terrific.
As you were conducting the research, can you remember any obstacles or challenges that you faced and how you overcame them?
I can say that the biggest challenge during the entire project was to show the re-endothelialization of the entire vessel after injury, including the adjacent uninjured segments. After trying several technical approaches, sometimes with pretty disappointing outcomes even following a huge amount of work, the best results were obtained by using scanning electron microscopy (thanks to the superb expertise of Dr. Kunihiro Uryo at Rockefeller University) and immunofluorescence of en face preparations of the arteries (Figure 1), which eventually turned out to be the most reliable techniques.
Were there any findings or results that you were not expecting at the outset of your work?
When we performed the assays on the platelets, using human blood samples to test platelet adhesion (by the way, we also used my blood for some experiments: I could actually see my own platelets running over the endothelial cells!), I have to confess that I didn’t expect the results that we obtained. They were so outstanding: basically, after treatment no platelets were attached in vitro to the surface mimicking the inner layer of the artery. It was so striking that it ultimately convinced me of the potential translational application of the work we were doing.
As a physician-scientist, how do you envision translating these findings into further preclinical models, clinical trials, and then further implementation into clinical practice?
This project is a proof-of-principle study, and there is undeniably so much to do in order to demonstrate the capacity to translate this project to a clinical setting. Dr. Totary-Jain is actively pursuing this in her new lab at the University of South Florida, and I am confident that with her enthusiasm and passion, this work can be advanced into the clinical scenario. Of course, I am always available in offering my expertise and open to collaborate.
Where are you now in your research career, and what are your future plans?
For now, I am just curious to know how things work and excited about discovering and learning the molecular mechanisms underlying some important human disorders. There is still a lot more research I want to do, and many questions need to be experimentally answered. I approach research by initially formulating a hypothesis, and then designing and performing experiments to test it. If I get good results, I apply for a grant and/or write a paper, but I also learned that negative results or mistakes are never useless. In fact they can open new theories and fields of investigation. To paraphrase William Blake, the true method of knowledge is experiment. So I feel it’s important to just experiment. I have big plans for my future, including the application for a career development award that will guarantee me the possibility to continue testing my scientific ideas, and I hope that this publication in the JCI will help me with that.
As a physician-scientist, how do you plan to balance the demands of teaching the next generation of researchers and clinicians, caring for patients, and doing research?
This is a great question. It’s difficult to answer. I believe that time management is the secret to balancing teaching, research, and clinical activity. As a postdoc, you are always more research focused, always concentrating day and night on thinking about new ideas and new experiments. As a physician-scientist, it’s even more complicated, since you have to consider both patients and scientific experiments. But in the end, as a matter of fact, I like it and I’ve always done that, so I think I will manage. My previous mentors in Italy (Dr. Guido Iaccarino and Dr. Bruno Trimarco) are physician-scientists, and here at Columbia Dr. Marks is a physician-scientist, so I am pretty sure to have the best advisors on my side!
Can you speak a little bit about the qualities of your experience in the lab with regard to what you think were the successful ingredients to make the final product a success?
Yes, I have to say that Dr. Marks’s laboratory at Columbia is certainly a unique environment where you have people with very different backgrounds and know-how working on various projects from calcium imaging to protein structure. So you rapidly grow up as an independent scientist and you have access to most of the literature in biomedical research. Having highly skilled people that are interested in your project and work with you day by day as friends is awesome. Dr. Totary-Jain, for instance, who led the project and recently established her independent lab at USF, is an expert in microRNA research and was always available to discuss all the aspects of the project, and together we overcame the main difficulties encountered in this study. Teaching young students like Melanie Gao to perform experiments, e.g., the vascular assays, was also a splendid experience. Having collaborations with scientists at different institutions (such as Dr. Thomas Tuschl of Rockefeller University) and in different departments at Columbia (such as Dr. Steven Marx) helped me to undertake this project. I received excellent suggestions from these collaborators in numerous meetings. When things weren’t going as well as I would have liked, there was encouragement from Drs. Totary-Jain and Marks. I really need to thank them for their support during this magnificent scientific journey.
(1) Santulli G, Iaccarino G, De Luca N, Trimarco B, Condorelli G. Atrial fibrillation and microRNAs. Front Physiol. 2014;5:15.
(2) Iaccarino G, et al. Ischemic neoangiogenesis enhanced by β2-adrenergic receptor overexpression: a novel role for the endothelial adrenergic system. Circ Res. 2005;97(11):1182–1189.
(3) Santulli G, et al. CaMK4 gene deletion induces hypertension. JAMA. 2012;1(4):e001081.
Gaetano Santulli, M.D., Ph.D., is currently a postdoctoral scientist in Dr. Andrew Marks’s laboratory at Columbia University Medical Center and trained as a cardiologist in Italy. He received his M.D. and his Ph.D. in clinical and experimental medicine from the University of Naples “Federico II” Medical School. He currently holds a postdoctoral fellowship from the American Heart Association. His research expertise is in hypertension, heart failure, aging, cardiac arrhythmias, angiogenesis, and metabolism.
Freddy T. Nguyen is an M.D./Ph.D. candidate at the University of Illinois at Urbana-Champaign. He is the Founder of the American Physician Scientists Association and served on the Associate Member Council of the American Association for Cancer Research. His research interests currently lie at the intersection of biomedical optics and cancer research. He received his B.S. in chemistry and B.A. in mathematics from Rice University.
Chirag Patel earned his M.D. and Ph.D. from the University of Texas Medical School at Houston. He completed a preliminary year of residency training in internal medicine at East Tennessee State University and is currently a resident physician in neurology at the University of California at Los Angeles David Geffen School of Medicine. Dr. Patel received his B.S. and M.S.E. in biomedical engineering from Johns Hopkins University.
Drugs currently approved to coat stents used in percutaneous coronary interventions do not discriminate between proliferating vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). This lack of discrimination delays reendothelialization and vascular healing, increasing the risk of late thrombosis following angioplasty. We developed a microRNA-based (miRNA-based) approach to inhibit proliferative VSMCs, thus preventing restenosis, while selectively promoting reendothelialization and preserving EC function. We used an adenoviral (Ad) vector that encodes cyclin-dependent kinase inhibitor p27Kip1 (p27) with target sequences for EC-specific miR-126-3p at the 3′ end (Ad-p27-126TS). Exogenous p27 overexpression was evaluated in vitro and in a rat arterial balloon injury model following transduction with Ad-p27-126TS, Ad-p27 (without miR-126 target sequences), or Ad-GFP (control). In vitro, Ad-p27-126TS protected the ability of ECs to proliferate, migrate, and form networks. At 2 and 4 weeks after injury, Ad-p27-126TS–treated animals exhibited reduced restenosis, complete reendothelialization, reduced hypercoagulability, and restoration of the vasodilatory response to acetylcholine to levels comparable to those in uninjured vessels. By incorporating miR-126-3p target sequences to leverage endogenous EC-specific miR-126, we overexpressed exogenous p27 in VSMCs, while selectively inhibiting p27 overexpression in ECs. Our proof-of-principle study demonstrates the potential of using a miRNA-based strategy as a therapeutic approach to specifically inhibit vascular restenosis while preserving EC function.
Gaetano Santulli, Anetta Wronska, Kunihiro Uryu, Thomas G. Diacovo, Melanie Gao, Steven O. Marx, Jan Kitajewski, Jamie M. Chilton, Kemal Marc Akat, Thomas Tuschl, Andrew R. Marks, Hana Totary-Jain